Vol 462 | 24/31 December 2009 | doi:10.1038/nature08588
ARTICLES
Structure of the outer membrane complex
of a type IV secretion system
Vidya Chandran1*, Rémi Fronzes1*, Stéphane Duquerroy2,3, Nora Cronin1, Jorge Navaza4 & Gabriel Waksman1
Type IV secretion systems are secretion nanomachines spanning the two membranes of Gram-negative bacteria. Three
proteins, VirB7, VirB9 and VirB10, assemble into a 1.05 megadalton (MDa) core spanning the inner and outer membranes.
This core consists of 14 copies of each of the proteins and forms two layers, the I and O layers, inserting in the inner and outer
membrane, respectively. Here we present the crystal structure of a ,0.6 MDa outer-membrane complex containing the
entire O layer. This structure is the largest determined for an outer-membrane channel and is unprecedented in being
composed of three proteins. Unexpectedly, this structure identifies VirB10 as the outer-membrane channel with a unique
hydrophobic double-helical transmembrane region. This structure establishes VirB10 as the only known protein crossing
both membranes of Gram-negative bacteria. Comparison of the cryo-electron microscopy (cryo-EM) and crystallographic
structures points to conformational changes regulating channel opening and closing.
Type IV secretion (T4S) systems are used by Gram-negative bacteria
in a variety of processes, ranging from the delivery of virulence factors
into eukaryotic cells to conjugative transfer of genetic material and
the uptake or release of DNA1–3. In Helicobacter pylori, Brucella suis
and Legionella pneumophila, T4S systems mediate the injection of
virulence proteins into mammalian host cells to cause gastric ulcers,
brucellosis, or Legionnaire’s disease, respectively4,5. In Agrobacterium
tumefaciens, the VirB/D T4S system delivers oncogenic DNA and
proteins into plant cells2,6, and Bordetella pertussis uses a T4S system
to secrete the pertussis toxin into the extracellular milieu7.
Conjugation promotes bacterial genome plasticity and the adaptive
response of bacteria to changes in the environment, contributing to
the spread of antibiotic-resistance genes among pathogenic bacteria8.
Although variations exist, many of the T4S systems found in
Gram-negative bacteria are similar to the A. tumefaciens VirB/D
T4S system, which comprises 12 proteins named VirB1 to VirB11
and VirD42. In general (but not always), T4S systems include an
extracellular pilus composed of a major (VirB2) and a minor
(VirB5) subunit9. Three ATPases located at the inner membrane,
VirB4, VirB11, and VirD4, power substrate secretion and possibly
assist in the assembly of the system2,10. The inner-membrane channel
is thought to be composed of the polytopic membrane protein VirB6
and the bitopic membrane proteins VirB8 and VirB102. At the outer
membrane, the composition of the pore that allows the substrate to
reach the extracellular milieu is unknown. VirB9 in complex with the
short lipoprotein VirB7 could be part of this structure. A region of the
carboxy (C)-terminal domain of VirB9 was shown to be surfaceexposed11. However, no transmembrane region could be found or
predicted in both proteins.
Recently, the cryo-EM structure of the core complex of the T4S
system encoded by the conjugative plasmid pKM101 showed that
T4S systems consist of a 1.05 MDa core spanning the inner and outer
membranes of Gram-negative bacteria12. This core, extracted preassembled from the membranes using detergents, is composed of
two layers, the O and I layers, and is formed by 14 copies of three
proteins, the homologues of the VirB7, VirB9 and VirB10 proteins,
termed TraN, TraO and TraF, respectively. Here we present the
crystal structure of the T4S system outer-membrane complex, containing the entire O layer.
General architecture of the complex
The T4S system outer-membrane complex was obtained from
chymotryptic cleavage of the T4S system core, crystallized and its
structure solved as described in the Methods (see also Supplementary Table 1 and Supplementary Fig. 1). This 590 kDa complex contains 14 copies each of the C-terminal domain of TraF (TraFCT;
residues 160–386), the C-terminal domain of TraO (TraOCT; residues 160–294) and the full-length TraN (Supplementary Fig. 1a). The
hetero-tetradecameric structure of the T4S system outer-membrane
complex has an elegant architecture with 14-fold symmetry (Fig. 1a).
In the cryo-EM work, the O layer was described as containing two
parts: the main body and the cap. In the crystal structure, the cap is
made of a hydrophobic ring of two-helix bundles defining a 32 Å
channel (Fig. 1a, b). These helices are made of residues 307–355 of
TraFCT. The main body is made of the rest of TraFCT, TraOCT and
TraN. It has a 172 Å diameter (Fig. 1b). When the complex is viewed
from the top, that is, from the extracellular milieu (Supplementary
Fig. 2a), TraFCT forms an inner ring surrounded by the TraOCT–
TraN complex. The TraN protein forms spokes radially crossing
the entire assembly (Supplementary Fig. 2a, b). When the complex
is viewed from the bottom, that is, from the periplasm (Supplementary Fig. 2c), the structure is primarily made of TraFCT. A cut-away
view of the structure (Fig. 1b) shows that TraF forms the entirety of
the inner wall of the structure, establishing VirB10 homologues as
T4S system outer-membrane channel proteins, an unexpected result
as VirB10 homologues have never been suspected to form the outermembrane channel of T4S systems. VirB10 homologues are also
found in the inner-membrane fraction owing to the presence of a
transmembrane helix at the amino (N) terminus of the protein13.
Thus, VirB10 is the only protein known to insert in both inner and
outer membranes of Gram-negative bacteria.
1
Institute of Structural and Molecular Biology, University College London and Birkbeck College, Malet Street, London WC1E 7HX, UK. 2Institut Pasteur, Unité de Virologie Structurale,
Virology Department and CNRS URA 3015, Paris, 25–28 Rue du Dr Roux, F-75724 Paris, France. 3Université Paris-Sud, F-91405 Orsay, France. 4Laboratoire de Microscopie
Electronique, Institut de Biologie Structurale J.P. Ebel, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France.
*These authors contributed equally to this work.
1011
©2009 Macmillan Publishers Limited. All rights reserved
ARTICLES
NATURE | Vol 462 | 24/31 December 2009
a
α2
Antenna
α3
b
Outer membrane
C
Cap 24 Å
Main
body
57 Å
β8
Periplasm
N
172 Å
N
Figure 1 | The T4S system outer-membrane complex. Ribbon diagram
(a) and space-filling cut-away (b) of the tetradecameric complex. TraFCT,
TraOCT and TraN subunits are colour-coded green, cyan and magenta,
respectively. In b, dimensions and labelling of the various parts of the
complex are provided.
Structure of the heterotrimer unit
A unique feature of the T4S system outer-membrane complex is that
it is made of three proteins (instead of one for all outer-membrane
structures previously determined), all three being indispensable for
complex assembly and channel formation12. The heterotrimer
formed by TraFCT, TraOCT and TraN is shown in Fig. 2 (see also Supplementary Fig. 2d). The interfaces between TraFCT and TraOCT and
between TraOCT and TraN bury 2861 Å2 and 2024 Å2 of surface area,
respectively. TraN does not interact with TraFCT in the heterotrimer.
The core structure of the TraOCT–TraN complex bound to TraFCT is
similar to the previously determined NMR structure of the TraOCT–
TraN complex alone (Fig. 2; details in Supplementary Information
and Supplementary Fig. 3a, b)11. However, there are also important
differences. First, in TraOCT bound to TraFCT, residues 162–173 at
the N terminus form the a1 helix (Fig. 2). In the tetradecamer, 14 a1
helices form 14 pillars onto which the entire complex sits on the I
layer (Fig. 1a). Second, at the C terminus, residues 286–289 have
become ordered in TraOCT bound to TraFCT to form the b10 strand.
b10 of TraOCT is part of the TraFCT–TraOCT interface as it forms a
two-stranded b-sheet with b8 of TraFCT (see Fig. 2 where, for clarity,
only b8 of TraFCT is indicated). In TraN, sequences at the N (residues
15–25) and C (residues 37–42) termini have become ordered in the
heterotrimer. Remarkably, TraN almost entirely wraps around
TraOCT, forming an interface with TraOCT that is much more extensive than that observed in the NMR structure (see details in Supplementary Information and Supplementary Fig. 3c).
The structure of the apo form of the C-terminal domain of
ComB10 (ComB10CT), a TraF/VirB10 homologue, has been solved14.
It consists of an atypical b-barrel flanked by an a-helix, onto which an
‘antenna’-like structure is mounted (Supplementary Fig. 3d). TraFCT
in the heterotrimer differs from ComB10CT in three major ways: (1)
the flanking helix of ComB10CT is missing in TraFCT; (2) the antenna
structure of TraFCT is more extended; and (3) the N terminus of
TraFCT forms an extended N-terminal arm (or lever arm; residues
N
C
α1
N-terminal arm
Figure 2 | Ribbon diagram of the heterotrimer unit. Structures mentioned
in the main text are indicated. The insert locates the shown heterotrimer
within the tetradecameric structure. A stereo version of this figure with full
secondary structure labelling is provided in Supplementary Fig. 2d.
171–199) that projects out to interact with three consecutive neighbouring heterotrimers in the tetradecameric assembly (Figs 2 and 3,
and Supplementary Figs 3e and 4).
Tetradecameric assembly
The interactions between heterotrimers in the tetradecameric outermembrane complex are extensive (details are provided in Supplementary Figs 4–7 and Supplementary Information). To help orientate the
reader, a consistent colour scheme summarized in Fig. 3a, e is provided,
in which the TraF/VirB10, TraO/VirB9 and TraN/VirB7 subunits are
numbered clockwise F1–F14, O1–O14 and N1–N14, respectively.
Of the 13,000 Å2 of total surface area of TraFCT, 11% is involved in
intra-heterotrimeric interactions and 51.5% is involved in interactions
between heterotrimers (Supplementary Fig. 8a, b). TraFCT of one
heterotrimer (for example F1 in green in Fig. 3a, b) interacts not only
with two adjacent TraFCT subunits (F2 and F14), but also, because of
the long N-terminal lever arm, with four neighbouring TraFCT subunits located further afield (F3, F4 on one side and F13 and F12 on the
other; Fig. 3b). In addition, TraFCT interacts with the TraOCT and TraN
subunits of an adjacent heterotrimer (O14 and N14 in Fig. 3a)
The interface between TraFCT subunits includes 2320 Å2 of surface
area and is described in detail in the Supplementary Information and
in Supplementary Fig. 6. Strand additions and numerous loop–loop
interactions constitute important parts of the interface. Yet, its most
notable feature is the N-terminal lever arm of the TraFCT subunit,
which makes its own extensive interaction network. The arm of subunit FX (1 # X # 14) interacts with the subunit FX 1 1, FX 1 2 and
FX 1 3 (for example, see Fig. 3b, c where the F1 lever arm interacts
with subunits F2, F3 and F4). FX 2 FX 1 1, FX 2 FX 1 2 and
FX 2 FX 1 3 contacts involve residues in the bn1 strand, the
bn1 2 an1 linker, and an1 helix of the FX subunit lever arm, respectively (Fig. 3c and Supplementary Fig. 6c, d). Overall, the N-terminal
lever arms form a continuous inner shelf at the base of the outermembrane complex (Fig. 3d).
1012
©2009 Macmillan Publishers Limited. All rights reserved
ARTICLES
NATURE | Vol 462 | 24/31 December 2009
F12
a
b
F13
F12
F13
O14
F14
F14
180º
F1
N14
O1
F2
F1
F3
F4
F2
F3
F4
c
d
F1
N
βn1
αn1
F2
F3
F4
e
f
F1
O14
F2
O1
O14
F1
N1
N1
F2
O1
O2
O2
Figure 3 | Inter-heterotrimeric interactions. a, Schematic diagram of the
tetradecamer with emphasis on interactions with the TraFCT subunit in green
(F1). TraFCT and TraOCT subunits are shown in circles and hexagons,
respectively. Not shown for clarity: TraN subunits (except N14, which makes
interactions with F1) and the lever arms of subunits not interacting with F1.
b, Ribbon diagram of the F1-interacting TraFCT subunits (that is, F4, F3, F2,
F14, F13 and F12) viewed from the periplasm; that is, turned 180u compared
with the view in a. The rectangle locates the lever arm of subunit F1.
c, Interactions of subunits F2, F3 and F4 with the lever arm of subunit F1. The
secondary structures of the lever arm of F1 are labelled. The F2, F3 and F4
subunits are in ribbon representation and labelled accordingly. d, Cut-away
side view of the outer-membrane complex with the proteins in ribbon
diagram representation colour-coded as in Fig. 1 but for the N-terminal arms
of the TraFCT subunits in red. e, Schematic diagram of the tetradecamer with
emphasis on interactions with the TraOCT subunit in cyan (O1). Not shown
for clarity: TraN subunits (except N1, which makes interactions with O1) and
the lever arms of subunits not interacting with O1. f, Ribbon diagram of the
subunits shown in colour at left. This view is from the extracellular milieu.
In the tetradecameric structure of the outer-membrane complex,
TraOCT interacts with five proteins: two adjacent TraOCT subunits, 1
TraFCT subunit of an adjacent heterotrimer, 1 TraFCT and 1 TraN
within its own heterotrimer (Fig. 3e, f). Overall, 49.25% of the total
surface area of TraOCT is involved in protein–protein interaction, 43%
of which is with the adjacent heterotrimer proteins (Supplementary
Fig. 8c, d). The interfaces between adjacent TraOCT subunits and
between TraOCT of one heterotrimer and TraFCT of an adjacent heterotrimer (described in the Supplementary Information and
Supplementary Fig. 7) mostly consist of loop residues (Fig. 3f).
panel) formed by helices a2 and a3 of the TraFCT antenna. These
helices each form a ring, with the a3 ring inside the a2 ring, suggesting
that a2 is the transmembrane helix contacting the membrane. Two
lines of evidence indicate that this region is inserted in the outer
membrane and forms the outer-membrane channel. First, a2, the
external helix in the two-helix bundle, has all the features of a transmembrane helix: it is amphipathic, with its hydrophobic side expected
to contact the membrane, and is strongly predicted to form a transmembrane helix by TMPred (http://www.ch.embnet.org/software/
TMPRED_form.html). Second, when a Flag-tag is inserted between
helices a2 and a3 of TraF in the full-length core complex, the tag is
found exposed extracellularly (Fig. 4b), demonstrating that the twohelix bundle projects across the outer membrane. Altogether, these
results unambiguously assign the two-helix bundle region of TraF/
VirB10 as the channel-forming region of T4S systems.
The outer-membrane pore
Viewed from the top, the structure contains a central hydrophobic ring
of 76 Å in diameter (Fig. 4a, left panel) with a 32 Å pore in the middle.
This region consists of a ring of two-helix bundles (Fig. 4a, lower right
1013
©2009 Macmillan Publishers Limited. All rights reserved
ARTICLES
NATURE | Vol 462 | 24/31 December 2009
a
b
76 Å
c
OM
N
N
IM
Figure 4 | Transmembrane region of the T4S system outer-membrane
complex and proposed mechanism of pore opening and closure. a, Left
panel: surface diagram of complex viewed from the extracellular milieu. The
two lines define the hydrophobic central region around the pore. Top right
panel: same as at left but showing (in yellow) the ring of Trp residues at the
base of a2. Bottom right panel: ribbon diagram of the transmembrane
helices with the internal and external ring of helices colour-coded in green
and red, respectively. b, Extracellular detection of the Flag-tag located
between helices a2 and a3 of TraF. A Flag-tag was introduced in the loop
between the a2 and a3 helices of TraF as described in Methods. Upper and
lower left panels: control sample expressing the wild-type T4S system core
complex. Upper and lower right panels: cells expressing the Flag-tagged T4S
system core complex. Differential interference contrast images are shown in
the upper panels and the corresponding fluorescence images in the lower
panels. c, Proposed mechanism for pore opening and closure. The cryo-EM
core complex structure (pale yellow) is superimposed on the crystal
structure of the outer-membrane complex (pale cyan). The N-terminal lever
arm shelf is highlighted in orange and red in the cryo-EM and crystal
structures, respectively. The position of the lipid on TraN/VirB7 is indicated
in black dots. TraN/VirB7 is shown in magenta. Proposed conformational
changes are illustrated by arrows. The N-terminal sequences linking the lever
arms to sequences in the I layer are shown in dashed lines colour-coded red
and orange for the conformations found in the crystal and cryo-EM
structures, respectively. OM, outer membrane; IM, inner membrane.
a-Helical insertions into outer membranes have been observed only
once before, in Wza15,16. In Wza, the C terminus forms an a-helix and
eight of them in the Wza octamer were shown unambiguously to form
the outer-membrane channel. The outer-membrane channel of the
T4S system differs by forming a two-helix bundle ring system. As in
Wza, a tryptophan residue lies at the N-terminal base of the TraF
transmembrane helix contacting the membrane (a2; Fig. 4a, upper
right panel)15. This Trp residue is conserved in most VirB10 protein
family members. In the structure presented here, the outer-membraneinserting region has been cleaved by chymotrypsin (Fig. 1a) and the
region around the cleavage is disordered (residues 322–344 could not
be traced because of poor electron density). As a result, the transmembrane a2 helices are one turn shorter than those forming the outermembrane channel of Wza. Another consequence of this structural
disorder is that the extent of the opening cannot be precisely defined.
The T4S system outer-membrane complex, in addition to being
much larger than previously described outer-membrane structures
(for example Wza or TolC), is also radically different in shape
(Supplementary Fig. 9): whereas the Wza homo-octamer and the
homo-TolC trimer are elongated15,17, the hetero-tetradecameric
T4S system outer-membrane complex spreads out just under the
outer membrane, creating a vast contact area with the inner leaflet
of the outer membrane. This contact area is mostly mediated by the
TraOCT–TraN complex ring. In effect, the TraOCT–TraN complex
ring appears to buttress the entire T4S system against the inner leaflet
of the outer membrane, possibly allowing the system to exert much
greater mechanical forces to extrude substrates than would otherwise
be feasible in systems like Wza or TolC, which make limited contacts
with the inner leaflet of the outer membrane.
Conformational changes in T4S systems
The cryo-EM structure of the T4S system core complex revealed a
double-walled structure in the cap of the O layer (Supplementary
Fig. 10a)12. The crystal structure of the cap is not double-walled.
This is because the lipid moiety of TraN/VirB7 was not built (the
single-wavelength anomalous dispersion (SAD)-derived map showed
electron density connecting to Cys 15 of TraN/VirB7 (the lipidation
site) but the density was not sufficiently well-defined for unambiguous
fitting). However, when the crystal structure of the outer-membrane
complex is superimposed on the cryo-EM structure of the entire complex (Fig. 4c), Cys 15 of TraN aligns perfectly at the base of the outer
wall of the cap, suggesting that this part of the cap might indeed
contain the lipidated part of TraN (indicated in Fig. 4c in black dots).
The superposition of the structure of the outer-membrane complex and that of the core complex (Fig. 4c; such a superposition is
valid as one complex is derived from the other by proteolysis) also
reveals that the transmembrane helices overlap partly with the first
half of the inner wall of the cap, suggesting that they might form this
region of the cap. However, the crystal structure captures them in a
different conformation than that observed in the cryo-EM structure:
whereas in the cryo-EM structure they form a narrow vertical constriction, in the crystal structure they lie in a ‘relaxed’ state at a 45u
1014
©2009 Macmillan Publishers Limited. All rights reserved
ARTICLES
NATURE | Vol 462 | 24/31 December 2009
angle. Thus, the two structures may represent different states.
Presumably, by removing the entire N-terminal half of TraF, the
constraints that this half places on the N-terminal lever arm of
TraFCT might have been removed, releasing the lever arms and leading to relaxation of the transmembrane helices. Indeed, as illustrated
in Fig. 4c, sequences N-terminal to the an1 helix would directly
connect to the I layer (schematically shown in Fig. 4c by dashed
red lines) and are likely to bring the N-terminal arm of each TraF/
VirB10 subunit down. This is consistent with the fact that the shelf
formed by the N-terminal lever arms of TraF/VirB10 subunits has
shifted up in the crystal structure compared with its position in the
cryo-EM structure of the full-length core complex (Fig. 4c and
Supplementary Fig. 10a, b in orange and red, respectively). Thus,
we propose that the N-terminal arms of the TraF/VirB10 subunits
might act in concert to exert conformational changes in the T4S
system channel upon signals sensed by the N-terminal domain of
the protein (see below).
In A. tumefaciens, VirB10 is known to undergo a conformational
change induced by the energizing T4S system components18. The
structure of the T4S system outer-membrane complex reveals that
VirB10 forms the outer-membrane channel. However, VirB10 is also
known to insert in the inner membrane, making contact not only with
the inner-membrane channel component VirB8, but also with the
ATPases13,19,20. Thus, VirB10 is in a unique position to relay conformational changes taking place in the ATPases and to effect pore opening and closure at the outer membrane. Also, in A. tumefaciens, VirB10
does not directly contact the substrate, but regulates its handover from
the VirB6/VirB8 complex in the inner membrane to VirB9 and VirB2,
the major pilin21. As VirB10 lines the interior of the outer-membrane
complex, it is difficult to envisage how the substrate could not interact
with it, unless it is insulated from the substrate by another layer of
protein, presumably made of the VirB2 pilin. We thus propose that the
VirB2 pilin forms a cylindrical conduit encased within the VirB10
ring. This hypothesis is also consistent with the observation that, in
the state captured in the crystal structure (where VirB2 is absent), the
TraF/VirB10 transmembrane helices have somewhat caved in.
The crystal structure of the T4S system outer-membrane complex
reveals an outer-membrane structure of unprecedented size and complexity. This structure is held together by a dense network of protein–
protein interactions, which provides a rich targeting ground for inhibitor design. Most striking among them is the extensive interaction that
the N-terminal lever arms of the TraFCT subunits make with numerous
subunits along the tetradecameric structure. We hypothesized that
these sequences are at the heart of a nanodevice regulating T4S. If
confirmed, this mechanism could become key to the design of inhibitor compounds specifically targeting the T4S machinery.
METHODS SUMMARY
Purification, crystallization, X-ray diffraction data collection. The T4S system
core complex was expressed and purified as described previously12. After addition of chymotrypsin, the T4S system outer-membrane complex was purified by
gel filtration. Crystals were grown by hanging-drop vapour diffusion. Native and
SAD data at the selenium edge were collected at the European Synchrotron
Radiation Facility beamline ID14.4 and Soleil’s beamline PROXIMA 1, respectively, and processed using the XDS package.
Structure determination and refinement. The structure was solved by molecular replacement using the native data set and the known 20 Å resolution cryo-EM
map of the trypsin-cleaved core complex as search model12. Fourteen-fold noncrystallographic symmetry averaging and phase extension to 2.8 Å resolution
yielded a readily interpretable electron density map. This map was used to locate
selenium atoms using the SAD data set. These heavy-metal sites were used to
generate SAD phases to 2.6 Å resolution, also yielding an interpretable electron
density map.
Extracellular localization of a Flag-tag inserted between the a2 and a3 helices
of TraF. A Flag-tag was introduced at position 332 in the loop between the a2
and a3 helices of TraF. Cells expressing either the wild-type core complex or the
Flag-tagged core complex were grown, fixed and incubated with anti-Flag antibodies followed by goat anti-mouse IgG1 Texas Red antibodies. Fluorescence
was monitored using a Zeiss Axioskop microscope, and images collected using a
Hamamatsu Orca ER camera.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 26 June; accepted 19 October 2009.
Published online 29 November 2009.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Fronzes, R., Christie, P. J. & Waksman, G. The structural biology of type IV
secretion systems. Nature Rev. Microbiol. 7, 703–714 (2009).
Christie, P. J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S. & Cascales, E.
Biogenesis, architecture, and function of bacterial type IV secretion systems.
Annu. Rev. Microbiol. 59, 451–485 (2005).
Schroder, G. & Lanka, E. The mating pair formation system of conjugative
plasmids – a versatile secretion machinery for transfer of proteins and DNA.
Plasmid 54, 1–25 (2005).
Backert, S. & Selbach, M. Role of type IV secretion in Helicobacter pylori
pathogenesis. Cell. Microbiol. 10, 1573–1581 (2008).
Ninio, S. & Roy, C. R. Effector proteins translocated by Legionella pneumophila:
strength in numbers. Trends Microbiol. 15, 372–380 (2007).
McCullen, C. A. & Binns, A. N. Agrobacterium tumefaciens and plant cell
interactions and activities required for interkingdom macromolecular transfer.
Annu. Rev. Cell Dev. Biol. 22, 101–127 (2006).
Burns, D. L. Type IV transporters of pathogenic bacteria. Curr. Opin. Microbiol. 6,
29–34 (2003).
Thomas, C. M. & Nielsen, K. M. Mechanisms of, and barriers to, horizontal gene
transfer between bacteria. Nature Rev. Microbiol. 3, 711–721 (2005).
Fronzes, R., Remaut, H. & Waksman, G. Architectures and biogenesis of non-flagellar
protein appendages in Gram-negative bacteria. EMBO J. 27, 2271–2280 (2008).
Gomis-Ruth, F. X. & Coll, M. Cut and move: protein machinery for DNA processing
in bacterial conjugation. Curr. Opin. Struct. Biol. 16, 744–752 (2006).
Bayliss, R. et al. NMR structure of a complex between the VirB9/VirB7 interaction
domains of the pKM101 type IV secretion system. Proc. Natl Acad. Sci. USA 104,
1673–1678 (2007).
Fronzes, R. et al. Structure of a type IV secretion system core complex. Science
323, 266–268 (2009).
Jakubowski, S. J. et al. Agrobacterium VirB10 domain requirements for type IV
secretion and T pilus biogenesis. Mol. Microbiol. 71, 779–794 (2009).
Terradot, L. et al. Structures of two core subunits of the bacterial type IV secretion
system, VirB8 from Brucella suis and ComB10 from Helicobacter pylori. Proc. Natl
Acad. Sci. USA 102, 4596–4601 (2005).
Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a
new class of membrane protein. Nature 444, 226–229 (2006).
Meng, G., Fronzes, R., Chandran, V., Remaut, H. & Waksman, G. Protein oligomerization in the bacterial outer membrane. Mol. Membr. Biol. 26, 136–145 (2009).
Koronakis, V., Sharff, A., Koronakis, E., Luisi, B. & Hughes, C. Crystal structure of
the bacterial membrane protein TolC central to multidrug efflux and protein
export. Nature 405, 914–919 (2000).
Cascales, E. & Christie, P. J. Agrobacterium VirB10, an ATP energy sensor required
for type IV secretion. Proc. Natl Acad. Sci. USA 101, 17228–17233 (2004).
Atmakuri, K., Cascales, E. & Christie, P. J. Energetic components VirD4, VirB11 and
VirB4 mediate early DNA transfer reactions required for bacterial type IV
secretion. Mol. Microbiol. 54, 1199–1211 (2004).
Llosa, M., Zunzunegui, S. & de la Cruz, F. Conjugative coupling proteins interact
with cognate and heterologous VirB10-like proteins while exhibiting specificity for
cognate relaxosomes. Proc. Natl Acad. Sci. USA 100, 10465–10470 (2003).
Cascales, E. & Christie, P. J. Definition of a bacterial type IV secretion pathway for
a DNA substrate. Science 304, 1170–1173 (2004).
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements This work was funded by Wellcome Trust grant 082227 to
G.W. We thank A. Thompson and the staff of beamline PROXIMA 1 at Soleil, the
staff of beamline ID14.4 at the European Synchrotron Radiation Facility, and
H. Saibil, E. Orlova and P. Christie for comments on the manuscript. We thank
A. Kumar for help in implementing the immunofluorescence experiments.
Author Contributions V.C. produced the complex, optimized crystals, and built,
refined and analysed the structure. R.F. designed the purification protocol,
produced the complex, grew the first crystals, optimized crystals and analysed the
structure. S.D. and J.N. solved the structure by molecular replacement and
provided the electron density map. N.C. collected crystallographic data. G.W.
supervised the work, analysed the structure and wrote the paper.
Author Information Structure factors and coordinates are deposited in the Protein
Data Bank under accession number 3JQO. Reprints and permissions information is
available at www.nature.com/reprints. Correspondence and requests for
materials should be addressed to G.W. ([email protected] or
[email protected]).
1015
©2009 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08588
METHODS
Purification of the outer-membrane complex. Expression of the T4S system
core complex from the IBA3c:traN-traFC-ST plasmid was induced using
200 mg l21 of anhydrotetracyclin12. After incubation overnight at 16 uC, the complex was extracted from the membrane fraction and purified using a Strep-Tactin
sepharose affinity column (IBA) as described previously12. The elution fractions
containing the core complex were pooled together and subjected to limited
proteolysis with 2 mg ml21 of chymotrypsin for 3 h at room temperature. The
proteolysed sample was then concentrated using a 100-kDa cut-off spin concentrator (Amicon) and loaded onto a Superose 6 GL 10/300 (GE Healthcare) gel
filtration column in 50 mM TrisHCl pH 8.0, 200 mM NaCl and 10 mM LDAO.
The outer-membrane complex eluted as a single peak (Supplementary Fig. 1a).
Selenomethionine labelling. The IBA3c:traN-traFC-ST plasmid12 was transformed into B834(DE3) competent cells (Novagen). Expression of the selenomethionine (SeMet)-labelled proteins was performed at 16 uC overnight in M9
minimal medium supplemented with 50 mg l21 of SeMet. Purification of the
SeMet-labelled outer-membrane complex was as described above for the native
complex.
Crystallization and data collection. Large rod-like crystals were grown by
vapour diffusion method at 20 uC using hanging drops containing 15 mg ml21
of the purified native and SeMet-labelled complexes and 20–40% w/v MPD,
100 mM Bis-Tris pH 6.5–7.5. The crystals were flash frozen in liquid nitrogen
using the mother liquor as the cryoprotectant. For the SeMet crystals, the oxidation protocol described in Sharff et al.22 was used before crystallization. The
oxidized SeMet crystals were further flash-soaked in 0.1% H2O2 in 50% w/v
MPD, 100 mM Bis-Tris, pH 7.0 before flash freezing for data collection. Data
collection was performed at the beamline ID14-4 at European Synchrotron
Radiation Facility (Grenoble, France) for the native data set and at beamline
PROXIMA 1 at the Soleil Synchrotron (Gif sur Yvette, France) for the oxidized
SeMet SAD data set. Data reduction was performed using the XDS package23.
Data collection statistics are presented in Supplementary Table 1.
Structure determination. The structure was solved by molecular replacement
using the native data set and the known 20 Å resolution cryo-EM map of the
trypsin-cleaved core complex as search model (Supplementary Fig. 1b)12. The
orientation and position of the particle was determined using AMoRe24. The
35–20 Å resolution range was used and yielded a molecular replacement solution
with a correlation coefficient of 0.39. Fourteenfold non-crystallographic symmetry averaging with RAVE, MAMA and other programs of the Uppsala suite25–27,
in combination with the CCP4 suite28, was used for phase extension to 2.8 Å
resolution using the native data set. This yielded a readily interpretable map with
well-defined side chains (Supplementary Fig. 1c and Supplementary Table 1). The
three component proteins TraFCT, TraOCT and TraN were traced manually with
the program Coot29. Subsequently, this structure was used to locate the selenium
atoms in a SAD data set collected to 2.6 Å resolution. Refinement of these sites and
phasing (PHASER30), followed by density modification exploiting the 14-fold
non-crystallographic symmetry in PARROT28, yielded a readily interpretable
electron density map. After initial tracing by BUCCANEER28, the partial model
was used for subsequent cycles of PHASER, PARROT and BUCCANNEER.
Further restrained refinement with REFMAC31 and PHENIX32,33 was performed
using tight restraints on non-crystallographic symmetry throughout the refinement. Final stages of refinement with the map from the SAD data set showed a
density for a modified cysteine in the lipoprotein TraN with small disordered lipid
chains attached. However, as the density was not good enough for unambiguous
building, we refrained from modelling this modification in the final model. The
final model has 94.8% residues in the most favoured region, 4.7% residues in the
additionally allowed regions and 0.5% residues in the disallowed region of the
Ramachandran plot. Refinement statistics are presented in the Supplementary
Table 1.
Extracellular localization of a Flag-tag inserted between the a2 and a3 helices of
TraF. The Flag epitope was introduced by PCR at position 332 of TraF/VirB10 in
the IBA3c:traN-traFC-ST plasmid12, yielding IBA3c:traN-traFFlag,C-ST. The primer
sequences were: 59-GACGACGACAAGATTCAGTACAACAGCACAGAA-39 (forward) and 59-GTCCTTGTAGTCGTTATTACTCTGCGTCTGGTT-39 (reverse).
Expression and purification of the resulting Flag-tagged T4S system core complex
yielded a complex very similar in molecular mass to the wild-type complex
(1.05 MDa), indicating that the Flag-tag does not disturb core complex assembly.
Escherichia coli TOP10 cells containing either the IBA3c:traN-traFC-ST plasmid (the
expression of which results in the production of the wild-type T4S system core
complex)12 or the IBA3c:traN-traFFlag,C-ST plasmid (the expression of which results
in the production of the T4S system core complex containing a Flag-tag at position
332 of TraF) were grown at 37 uC to an optical density (OD600 nm) of 0.6. Protein
expression was induced by addition of 200 mg l21 of anhydrotetracyclin and cells
were incubated at 16 uC overnight. Cells were collected by centrifugation, resuspended in ice-cold 4% formaldehyde and incubated on ice for 15 min. The fixed
cells were washed twice with PBS and re-suspended in PBS. The cell suspensions
were applied to poly-L-lysine-treated microscope slides and incubated at room
temperature for 20 min. The slides were rinsed in PBS and blocked in 1% goat
serum-PBS for 45 min. The primary antibody (anti-Flag M2 Mab (Sigma), 1:1,000
dilution) was applied and incubated on the slide at 4 uC overnight. The slides were
washed twice with PBS at room temperature and the secondary antibody (goat antimouse IgG1 Texas Red (Southern Biotech), 1:500 dilution) was applied and incubated on the slide for 1 h. The slides were then washed twice with PBS and were
mounted using DAKO fluorescent mounting medium. The fluorescence microscopy was performed on a Zeiss Axioskop microscope, objective 3100 with oil. The
images were collected using a Hamamatsu Orca ER camera.
22. Sharff, A. J., Koronakis, E., Luisi, B. & Koronakis, V. Oxidation of selenomethionine:
some MADness in the method! Acta Crystallogr. D 56, 785–788 (2000).
23. Kabsch, W. Automatic processing of rotation diffraction data from crystals of
initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800
(1993).
24. Navaza, J. AMoRe: an automated package for molecular replacement. Acta
Crystallogr. A 50, 157–163 (1994).
25. Jones, T. A. in Molecular Replacement 91–95 (CCP4 Proceedings, 1992) Æhttp://
epubs.cclrc.ac.uk/bitstream/948/DL-SCI-R33.pdfæ.
26. Kjeldgaard, M. & Jones, T. A. in From First Map to Final Model 59–66 (CCP4
Proceedings, 1994) Æhttp://epubs.cclrc.ac.uk/bitstream/950/DL-SCI-R35.pdfæ.
27. Kleywegt, G. & Jones, T. Software for handling macromolecular envelopes. Acta
Crystallogr. D 55, 941–944 (1999).
28. Collaborative Computational Project, Number 4. The CCP4 suite: programs for
protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
29. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta
Crystallogr. D 60, 2126–2132 (2004).
30. McCoy, A. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674
(2007).
31. Murshudov, G., Vagin, A. & Dodson, E. Refinement of macromolecular structures
by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).
32. Adams, P. D. et al. PHENIX: building new software for automated crystallographic
structure determination. Acta Crystallogr. D 58, 1948–1954 (2002).
33. Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. The Phenix refinement
framework. CCP4 Newsl. 42, contribution 8 (2005) Æhttp://www.ccp4.ac.uk/
newsletters/newsletter42.pdfæ.
©2009 Macmillan Publishers Limited. All rights reserved